Preparation process of anti-interference urine trace protein and urine creatinine double-chromogenic detection reagent

Abstract

The invention relates to the technical field of biological detection reagents, in particular to a preparation process of an anti-interference urine trace protein and urine creatinine double-chromogenic detection reagent, which comprises the following steps: dissolving a composite buffer agent in deionized water at 20-38 DEG C, stirring and dissolving, and adjusting the pH value of the system to 7.0-7.3; adding a composite anti-interference agent, an EDTA-2Na masking agent and enzymes in sequence, continuously stirring, and controlling the temperature of the system not to exceed 38 DEG C; independently preparing an improved BCA color developing agent and a copper sulfate auxiliary color developing agent, mixing and standing; adding the color developing system into the buffer solution system, adding a compound stabilizer, stirring and standing; by adopting a multi-enzyme synergistic composite anti-interference system and combining the synergistic effect of the masking agent, the influence of various interfering substances in urine can be effectively shielded, and non-specific reaction between the interfering substances and a detection system is avoided.

Description

Technical Field

[0001] This invention relates to the field of biological detection reagent technology, specifically to a preparation process for an interference-resistant dual-colorimetric detection reagent for urinary microprotein and urinary creatinine. Background Technology

[0002] The combined detection of urinary microalbumin and urinary creatinine is a crucial means of clinically assessing kidney function and detecting kidney damage at an early stage. The accuracy of the test results directly affects the formulation of disease diagnosis and treatment plans. With the continuous improvement of clinical testing needs, higher requirements are being placed on the comprehensive performance of testing reagents.

[0003] Existing testing reagents still have many limitations in practical applications. Insufficient interference resistance is a prominent problem; substances such as vitamins, bilirubin, hemoglobin, uric acid, and glucose in urine samples can easily interact with the detection system, leading to deviations in test results. Inadequate buffer system design can cause pH fluctuations under different temperature conditions, thus affecting enzyme activity and the stability of the colorimetric reaction.

[0004] The formulation of colorimetric systems lacks optimization, and some reagents exhibit narrow linear ranges, making them unsuitable for detecting samples of varying concentrations. Furthermore, the preparation processes are relatively crude, lacking targeted optimization measures, such as ineffective degassing and improper dispensing and sealing methods. This leads to performance degradation during storage, with poor stability under room temperature, refrigeration, and repeated freeze-thaw cycles. In addition, the precision and accuracy of existing reagents are insufficient to meet the requirements of high-precision clinical testing; some products exhibit high coefficients of variation and recoveries deviating from the ideal range, limiting their reliability in clinical diagnostics. Therefore, developing a dual-colorimetric detection reagent with strong anti-interference capabilities, good stability, and high detection accuracy, along with its preparation process, has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The primary objective of this invention is to provide a preparation process for an interference-resistant dual-colorimetric detection reagent for urinary microprotein and urinary creatinine, comprising the following steps: (1) Buffer preparation: Dissolve the composite buffer in deionized water at 20-38℃ and stir to dissolve, and adjust the pH of the system to 7.0-7.3; (2) Addition of functional components: Add the compound anti-interference agent, EDTA-2Na masking agent and enzymes in sequence, stir continuously, and control the system temperature to not exceed 38℃; (3) Preparation of colorimetric system: Prepare the modified BCA colorimetric agent and copper sulfate auxiliary colorimetric agent separately, mix them and let stand; (4) Combination and optimization: Add the colorimetric system to the buffer system, add the combination stabilizer, stir and let stand; (5) Purification and dispensing: Before filtration, ultrasonic degassing is performed. After filtration, the product is aseptically dispensed, vacuum sealed, and then refrigerated for later use. The composite anti-interference agent contains at least two of ascorbic acid oxidase, bilirubin oxidase, and hemoglobin oxidase. The composite buffer is a combination of two different buffers.

[0006] Preferably, the composite anti-interference agent is a combination of ascorbic acid oxidase and bilirubin oxidase, a combination of bilirubin oxidase and hemoglobin oxidase, or a combination of ascorbic acid oxidase, bilirubin oxidase and hemoglobin oxidase.

[0007] Preferably, the composite buffer is a combination of Tris-HCl and HEPES, or a combination of HEPES and PIPES, and the molar ratio of the two buffers in the combination is 3:2 to 7:3.

[0008] Preferably, the enzymes include sarcosine oxidase and peroxidase, with sarcosine oxidase used at a concentration of 0.15-0.2 g / L and peroxidase used at a concentration of 0.2-0.25 g / L.

[0009] Preferably, the volume ratio of the modified BCA colorimetric agent to the copper sulfate auxiliary colorimetric agent in the colorimetric system is 6:1 to 11:1.

[0010] Preferably, the composite stabilizer is a combination of glycerol and bovine serum albumin, a combination of glycerol and sorbitol, or a combination of bovine serum albumin and trehalose.

[0011] Preferably, the ultrasonic degassing treatment has a power of 100W and a treatment time of 5-10 minutes.

[0012] Preferably, the stirring speed in step 2 is 200-250 r / min, and the stirring time is 30-45 min.

[0013] Preferably, the settling time of the color development system in step 3 is 1.5-2 hours, and the settling time of the composite system in step 4 is 1.5-2.5 hours.

[0014] Preferably, the filter membrane used for filtration is a polyethersulfone filter membrane, and the refrigeration temperature after vacuum sealing is 2-6℃.

[0015] Preferably, the filter membrane is a polyethersulfone filter membrane, and the refrigeration temperature after vacuum sealing is 4°C.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs a multi-enzyme synergistic composite anti-interference system, combined with the synergistic effect of masking agents, to effectively shield the influence of various interfering substances in urine, avoid non-specific reactions between interfering substances and the detection system, ensure the authenticity of detection results in complex sample environments, and solve the problems of narrow anti-interference spectrum and susceptibility to impurities in existing reagents.

[0017] 2. The optimized composite buffer system in this invention effectively stabilizes the pH value of the detection environment, resists the adverse effects of temperature changes, and provides stable conditions for enzyme activity and colorimetric reactions. Simultaneously, the rationally proportioned enzyme and colorimetric system broadens the linear detection range, improves the correlation coefficient, significantly reduces the coefficient of variation of the detection results, and brings the recovery rate closer to the theoretical value, ensuring the accuracy and reliability of detection results for samples of different concentrations.

[0018] 3. This invention significantly improves the storage stability of reagents by introducing a composite stabilizer combination and employing refined preparation processes such as ultrasonic degassing and vacuum sealing. Whether stored at room temperature, under long-term refrigeration, or subjected to repeated freeze-thaw cycles, the degradation of reagent performance is greatly reduced, extending the product's shelf life, ensuring consistent performance during clinical use, and minimizing detection errors caused by reagent instability.

[0019] 4. The process steps are designed in a coherent manner, and the parameters at each stage are optimized and adapted, effectively controlling product quality and ensuring consistency and stability in batch production. The prepared reagents are compatible with fully automated biochemical analyzers, meeting the high-efficiency needs of clinical batch testing, and have broad clinical application prospects and promotional value. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] The general preparation process in the embodiments of the present invention is as follows: (1) Buffer preparation: Dissolve the buffer in deionized water at 20-38℃, stir at 80-350r / min for 8-35min, and adjust the pH to the target value; (2) Addition of functional components: Add the compound anti-interference agent, masking agent and enzyme in sequence, stir continuously for 15-65 min, and control the system temperature to not exceed 38℃; (3) Preparation of colorimetric system: Prepare the modified BCA colorimetric agent and auxiliary colorimetric agent separately, mix them and let stand for 0.8-3.5h; the modified BCA colorimetric agent is prepared by dissolving 1.0g bicinchoninic acid, 10g sodium carbonate, 5g sodium bicarbonate, 2g sodium hydroxide and 0.5g potassium sodium tartrate in deionized water, and the protein binding specificity is improved by optimizing the ratio of ligands; (4) Combination and optimization: Add the colorimetric system to the buffer system, add the stabilizer, stir at 80-350 r / min for 25-65 min, and let stand for 0.8-3.5 h; (5) Purification and dispensing: Filter through a 0.22μm filter membrane. Before filtration, ultrasonic degassing can be performed. After sterile dispensing, vacuum seal and store at 2-8℃ for later use.

[0022] All the following examples are prepared using a 1L system, and only the specific parameters are presented: Example 1: An interference-resistant dual-colorimetric reagent for detecting urine microalbumin / creatinine comprises the following components: Reagent 1 components: Tris-HCl buffer 0.1 mol / L (pH 7.0), sarcosine oxidase 0.2 g, horseradish peroxidase (model P8375, purchased from Sigma-Aldrich) 0.2 g, compound anti-interference agent (ascorbic acid oxidase) 0.3 g, masking agent EDTA-2Na 0.1 g, stabilizer (glycerol) 1.0 g, deionized water to a final volume of 1 L; Reagent 2 components: HEPES buffer 0.1 mol / L (pH 7.2), modified BCA chromogenic reagent 200 mL (prepared by dissolving 1.0 g bicinchoninica acid, 10 g sodium carbonate, 5 g sodium bicarbonate, 2 g sodium hydroxide, and 0.5 g potassium sodium tartrate in deionized water), auxiliary chromogenic reagent (copper sulfate solution, concentration 0.05 mol / L) 20 mL, compound anti-interference agent (bilirubin oxidase) 0.2 g, stabilizer (bovine serum albumin) 0.5 g, and deionized water to a final volume of 1 L; The preparation steps of this dual-colorimetric detection reagent are as follows: at 28℃, operate according to the general process, with a stirring speed of 200r / min, a buffer solution dissolution time of 20min, a functional component stirring time of 30min, a colorimetric system standing time of 1.5h, a compounding stirring time of 30min, a standing time of 1.5h, filtered through a 0.22μm filter membrane, dispensed, and stored at 4℃ for later use.

[0023] Example 2: Based on Example 1, the anti-interference system and auxiliary component allocation ratio were optimized: Reagent 1 components: Tris-HCl buffer 0.1 mol / L (pH 7.0), sarcosine oxidase 0.2 g, horseradish peroxidase (model P8375, purchased from Sigma-Aldrich) 0.2 g, compound anti-interference agent (ascorbic acid oxidase 0.2 g + bilirubin oxidase 0.2 g), masking agent EDTA-2Na 0.15 g, stabilizer (glycerol) 1.0 g, deionized water to a final volume of 1 L; The reagent consists of two components: 0.1 mol / L HEPES buffer (pH 7.2), 200 mL of modified BCA chromogenic reagent (prepared by dissolving 1.0 g bicinchoninica, 10 g sodium carbonate, 5 g sodium bicarbonate, 2 g sodium hydroxide, and 0.5 g potassium sodium tartrate in deionized water), 20 mL of auxiliary chromogenic reagent (copper sulfate solution, concentration 0.05 mol / L), a compound anti-interference agent (0.15 g bilirubin oxidase + 0.15 g hemoglobin oxidase), a stabilizer (0.8 g glycerol + 0.3 g bovine serum albumin), and deionized water to a final volume of 1 L. Preparation steps: At 28℃, first add the composite anti-interference agent and stir at 200r / min for 30min, then add the masking agent and enzymes, and then follow the steps in Example 1. The buffer solution dissolution time is 20min, the functional component stirring time is 30min, the color development system standing time is 1.5h, the composite stirring time is 30min, the standing time is 1.5h, the mixture is filtered through a 0.22μm filter membrane, dispensed and stored at 4℃ for later use.

[0024] This embodiment optimizes the anti-interference system through multi-enzyme combination, adds hemoglobin oxidase to improve the interference shielding range, and adjusts the ratio of masking agent and stabilizer, significantly improving the broad-spectrum anti-interference performance.

[0025] Example 3: Based on the optimized buffer system and colorimetric ratio of Example 2: Reagent 1 components: complex buffer (Tris-HCl 0.08mol / L + HEPES 0.06mol / L, pH 7.1), sarcosine oxidase 0.15g, horseradish peroxidase (model P8375, purchased from Sigma-Aldrich) 0.25g, complex anti-interference agent (ascorbic acid oxidase 0.2g + bilirubin oxidase 0.2g), masking agent EDTA-2Na 0.15g, stabilizer (glycerol) 1.2g, deionized water to a final volume of 1L; The reagent consists of two components: a complex buffer (HEPES 0.07 mol / L + PIPES 0.05 mol / L, pH 7.3), 180 mL of modified BCA chromogenic reagent (prepared by dissolving 1.0 g of bicinchoninica, 10 g of sodium carbonate, 5 g of sodium bicarbonate, 2 g of sodium hydroxide, and 0.5 g of potassium sodium tartrate in deionized water), 30 mL of auxiliary chromogenic reagent (copper sulfate solution, concentration 0.05 mol / L), a complex anti-interference agent (bilirubin oxidase 0.15 g + hemoglobin oxidase 0.15 g), a stabilizer (bovine serum albumin) 0.6 g, and deionized water to a final volume of 1 L. Preparation steps: At 30℃, first add the composite anti-interference agent and stir at 250r / min for 30min, then add the masking agent and enzymes. Let the color development system stand for 2h, stir for 30min after compounding, and let stand for 2h. Before filtration, add ultrasonic degassing treatment (power 100W, time 5min), filter through a 0.22μm filter membrane, dispense and store at 4℃ for later use.

[0026] This embodiment uses a composite buffer to improve pH stability, adjusts the ratio of enzymes to chromogenic agents, and adds an ultrasonic degassing step to reduce bubble interference, thus significantly improving buffer stability and chromogenic sensitivity.

[0027] Example 4: Based on Example 3, the buffer concentration, anti-interference agent ratio, and preparation process were optimized: Reagent 1 components: complex buffer (Tris-HCl 0.12 mol / L + HEPES 0.06 mol / L, pH 7.0), sarcosine oxidase 0.2 g, horseradish peroxidase (model P8375, purchased from Sigma-Aldrich) 0.2 g, complex anti-interference agent (ascorbic acid oxidase 0.25 g + bilirubin oxidase 0.25 g + hemoglobin oxidase 0.1 g), masking agent EDTA-2Na 0.2 g, stabilizer (glycerol 1.5 g + sorbitol 0.5 g), deionized water to a final volume of 1 L; The reagent consists of two components: a complex buffer (HEPES 0.1 mol / L + PIPES 0.06 mol / L, pH 7.2), 220 mL of modified BCA chromogenic reagent (prepared by dissolving 1.0 g of bicinchoninica, 10 g of sodium carbonate, 5 g of sodium bicarbonate, 2 g of sodium hydroxide, and 0.5 g of potassium sodium tartrate in deionized water), 22 mL of auxiliary chromogenic reagent (copper sulfate solution, concentration 0.05 mol / L), a complex anti-interference agent (bilirubin oxidase 0.15 g + hemoglobin oxidase 0.15 g), a stabilizer (bovine serum albumin 0.8 g + trehalose 0.4 g), and deionized water to a final volume of 1 L. Preparation steps: At 30℃, first add the composite anti-interference agent and stir at 250r / min for 45min, then add the masking agent and enzymes. Let the color development system stand for 2h, stir for 30min after compounding, and let stand for 2.5h. Before filtration, add ultrasonic degassing treatment (power 100W, time 5min), filter through a 0.22μm polyethersulfone filter membrane, dispense and vacuum seal, and store at 4℃ for later use.

[0028] In this embodiment, the buffer concentration was increased to enhance buffering capacity, the ratio of compound anti-interference agents was optimized, sorbitol and trehalose were introduced as low-temperature stabilizers, and the filtration and dispensing processes were optimized, resulting in a significant improvement in the long-term stability and repeated freeze-thaw tolerance of the reagents.

[0029] Example 5: By combining the optimization directions of Examples 1-4, and balancing anti-interference, detection accuracy, and stability, the optimal formula and process were determined: Reagent 1 components: complex buffer (Tris-HCl 0.1mol / L + HEPES 0.05mol / L, pH 7.0), sarcosine oxidase 0.18g, horseradish peroxidase (model P8375, purchased from Sigma-Aldrich) 0.22g, complex anti-interference agent (ascorbic acid oxidase 0.2g + bilirubin oxidase 0.2g + hemoglobin oxidase 0.15g), masking agent EDTA-2Na 0.18g, stabilizer (glycerol 1.2g + sorbitol 0.3g), deionized water to a final volume of 1L; The reagent consists of two components: a compound buffer (HEPES 0.08 mol / L + PIPES 0.04 mol / L, pH 7.2), 200 mL of modified BCA chromogenic reagent (prepared by dissolving 1.0 g of bicinchoninica, 10 g of sodium carbonate, 5 g of sodium bicarbonate, 2 g of sodium hydroxide, and 0.5 g of potassium sodium tartrate in deionized water), 25 mL of auxiliary chromogenic reagent (copper sulfate solution, concentration 0.05 mol / L), a compound anti-interference agent (bilirubin oxidase 0.15 g + hemoglobin oxidase 0.15 g), a stabilizer (bovine serum albumin 0.6 g + trehalose 0.3 g), and deionized water to a final volume of 1 L. Preparation steps: At 29℃, add the following components in the following order: buffer solution, composite anti-interference agent, masking agent, enzyme, colorimetric system, and stabilizer. Stir at 220 r / min at each stage, stir for 30 min for the functional components, let the colorimetric system stand for 2 h, stir for 30 min after compounding, let stand for 2 h, degas with ultrasound (100 W power, 5 min), filter through a 0.22 μm polyethersulfone membrane, vacuum seal, and store at 4℃ for later use.

[0030] This embodiment can achieve synergistic adaptation of the formula and process, and achieves optimal results in terms of anti-interference, detection accuracy and stability, which can meet the needs of high-precision clinical testing.

[0031] Comparative Example 1: The formulation and preparation steps are the same as in Example 2, except that the composite anti-interference agent is replaced with 0.4g of single ascorbic acid oxidase, while the dosage of other components and process parameters remain unchanged (the peroxidase is horseradish peroxidase, model P8375).

[0032] Comparative Example 2: The formulation and preparation steps are the same as in Example 3, except that the complex buffer of Reagent 1 is replaced with a single Tris-HCl buffer of 0.14 mol / L (pH 7.1), and the complex buffer of Reagent 2 is replaced with a single HEPES buffer of 0.12 mol / L (pH 7.3). The amounts of other components and process parameters remain unchanged (the peroxidase is horseradish peroxidase, model P8375).

[0033] Comparative Example 3: The formulation and preparation steps are the same as in Example 4, except that the ratio of the modified BCA chromogenic agent to the auxiliary chromogenic agent in reagent 2 is adjusted to 1:3 (i.e., 300mL of chromogenic agent + 100mL of auxiliary chromogenic agent). The dosage of other components and process parameters remain unchanged (the peroxidase is horseradish peroxidase, model P8375).

[0034] Comparative Example 4: The formula is the same as in Example 5. The preparation steps adopt the existing traditional process: no ultrasonic degassing step, stirring speed 100 r / min, stirring time of functional components 30 min, color development system and standing time after compounding are both 30 min, filtration is carried out using a 0.45 μm filter membrane, no vacuum sealing after dispensing, and other parameters remain unchanged (the peroxidase is horseradish peroxidase, model P8375).

[0035] Comparative Example 5: The formulation and preparation steps are the same as in Example 5, except that the EDTA-2Na masking agent in reagent 1 is removed, while the amounts of other components and process parameters remain unchanged (the peroxidase is horseradish peroxidase, model P8375).

[0036] Comparative Example 6: This comparative example is a simple combination of existing technologies. The formulation combines the composite anti-interference system of Example 2 with the single buffer system of Example 2. The process adopts the traditional steps of Example 1 (without ultrasonic degassing and vacuum sealing). The dosage of other components is the same as that of Example 2. The preparation steps are the same as those of Example 1 (wherein the peroxidase is horseradish peroxidase, model P8375).

[0037] Test samples and instruments: (1) Test samples: Reagents prepared in Examples 1-5 and Comparative Examples 1-6, with 3 copies of each sample prepared in parallel; (2) Testing instruments: fully automated biochemical analyzer, pH meter (accuracy 0.01), ultraviolet-visible spectrophotometer (wavelength range 400-800nm), constant temperature incubator (accuracy ±0.5℃); (3) Test urine samples: blank urine samples from healthy individuals, simulated urine samples with different concentrations of interfering substances (interfering substances include vitamin C 100-500 mg / L, bilirubin 10-50 mg / L, hemoglobin 50-250 mg / L, uric acid 500-2500 mg / L, glucose 10-50 g / L), and clinically positive urine samples (urine microprotein 200 mg / L, urine creatinine 500 μmol / L).

[0038] Test items and methods: (1) Linear range test: Prepare standard solutions of urine microprotein concentration gradient (5, 10, 50, 100, 200, 300, 400, 500 mg / L) and urine creatinine concentration gradient (10, 50, 100, 200, 300, 500, 800, 1000 μmol / L), test each reagent separately, record the absorbance value, plot the standard curve, calculate the correlation coefficient R², and determine the linear range.

[0039] (2) Precision test: Take clinical positive urine samples, repeat the test 10 times with each reagent, and calculate the mean (x), standard deviation (SD) and coefficient of variation (CV) of the urine microprotein and urine creatinine test results. CV≤2.0% is qualified.

[0040] (3) Accuracy test: The spiked recovery method was used. 100 mg / L of urine microprotein standard and 300 μmol / L of urine creatinine standard were added to the blank urine sample. Each reagent was tested separately and the recovery rate was calculated (recovery rate = measured value / theoretical value × 100%). A recovery rate of 95%-105% is considered qualified.

[0041] (4) Interference resistance test: Different concentrations of interfering substances were added to the clinical positive urine samples, and each reagent was used for testing. The interference rate was calculated (interference rate = (detection value after adding interfering substance - detection value without adding interfering substance) / detection value without adding interfering substance × 100%). An interference rate of ≤3.0% is considered qualified.

[0042] (5) Stability test: Room temperature stability: Each reagent was placed in a constant temperature incubator at 25℃, and the linear range, precision and accuracy were tested at 0, 1, 2, 3 and 6 months, and the performance degradation was recorded; Cold storage stability: Each reagent was placed in a refrigerated container at 4℃, and the above indicators were tested at 0, 3, 6, 9 and 12 months; Repeated freeze-thaw stability: Each reagent was frozen at -20℃ and thawed at room temperature, repeated 5 times, and the performance indicators after freeze-thaw were tested.

[0043] (6) pH stability test: each reagent is placed in an environment of 4℃, 25℃ and 37℃ for 7 days. The pH value of reagent 1 and reagent 2 is tested every day and the pH change range is recorded. The change range ≤0.1 is qualified.

[0044] Test Results and Analysis: Table 1. Linear Range Test Results

[0045] The results showed that Examples 1-5 had a wide linear range and high correlation coefficients. Comparative Example 3 had a significantly narrowed linear range due to the deviation of the colorimetric reagent ratio from the protected range. The linear range of Comparative Example 6 was still inferior to that of the present invention, indicating that the formulation and process design of the present invention can effectively ensure wide linear adaptability.

[0046] Precision testing uses the coefficient of variation (CV) as the core indicator. A CV of ≤2.0% is considered acceptable. The results are shown in Table 2 below: Table 2 Precision Test Results

[0047] The results showed that the coefficients of variation for Examples 1-5 were all ≤1.5%, with Example 5 exhibiting the best precision. The CV values ​​for Comparative Examples 2, 4, and 6 all exceeded the acceptable threshold, indicating that the composite buffer system and refined process of the present invention can significantly improve the detection precision.

[0048] Table 3 Accuracy Test Results

[0049] The results showed that the recovery rates of Examples 1-5 were all in the excellent range of 96%-104%, with the recovery rate of Example 5 being closest to the theoretical value. Comparative Examples 1 and 5 had unsatisfactory accuracy due to imperfect anti-interference systems, and the accuracy of Comparative Example 6 was still lower than that of the present invention. This indicates that the synergistic effect of the composite anti-interference system and the masking agent of the present invention can effectively improve the detection accuracy.

[0050] Table 4. Results of Anti-interference Test

[0051] For the anti-interference test, high-concentration interferents (vitamin C 500 mg / L, bilirubin 50 mg / L, uric acid 2000 mg / L) were selected. The interference rate ≤ 3.0% was considered qualified. The results showed that the interference rates of Examples 3-5 under high-concentration interferents were all ≤ 2.5%, indicating excellent anti-interference effects. Comparative Example 1, Comparative Example 5, and Comparative Example 6 were all unqualified due to imperfect anti-interference systems or insufficient synergy, demonstrating that the multi-enzyme synergistic composite anti-interference system and masking agent of the present invention can effectively shield various interferents.

[0052] Table 5 Results of Stability Test

[0053] The results showed that the room temperature stability, cold storage stability, and repeated freeze-thaw stability of Examples 1-5 were all better than those of the comparative examples: the attenuation rate of Example 5 at room temperature for 6 months was ≤ 4.0%, the attenuation rate at cold storage for 12 months was ≤ 5.0%, and the attenuation rate after repeated freeze-thaw for 5 times was ≤ ......

[0054] Table 6 Results of pH Stability Test

[0055] The results showed that the pH change ranges of Examples 1-5 in different temperature environments all met the qualified standards, and Examples 3-5 had the best pH stability; the pH change ranges of Comparative Example 2 and Comparative Example 6 exceeded the threshold, indicating that the composite buffer system of the present invention can significantly improve pH stability and adapt to the detection requirements at different temperatures.

[0056] The gradient optimization of the examples of the present invention verified the relevance and rationality of each technical solution, and the expanded raw material ratio and process parameter range provided strong support for technical protection. The performance test showed that the reagent of the present invention performed excellently in terms of anti-interference, stability, detection accuracy, etc.

[0057] In addition, it should be noted that the composite anti-interference agent refers to a combination of at least two oxidases. Ascorbic acid oxidase, bilirubin oxidase, and hemoglobin oxidase specifically degrade vitamin C, bilirubin, and hemoglobin in urine, respectively, while EDTA-2Na masking agent chelates metal ions, forming a dual anti-interference mechanism of "enzyme degradation and masking agent chelation," which covers 2-3 times more types of interfering substances than a single anti-interference agent. The modified BCA chromogenic agent improves binding specificity by optimizing the ratio of ligands, and its stability time is improved by more than 30% compared to traditional products. The composite buffer uses a combination of buffer ranges that are highly matched with the pH of the detection system. Tris-HCl provides strong buffer capacity, HEPES optimizes temperature stability, and PIPES enhances the ability to resist ionic strength interference. The molar concentration ratio of 3:2 to 7:3 ensures that the pH change of the system is ≤0.1 at 4-37℃, which is more than 50% lower than that of a single buffer.

[0058] Its core technical mechanism lies in the synergistic effect of the composite anti-interference system and masking agent, the stable pH environment of the composite buffer system, and the colorimetric system with a volume ratio of 6:1 to 11:1 to improve complexation efficiency. These three factors work together to ensure the accuracy of detection. Meanwhile, the ultrasonic degassing with a power of 100W and a duration of 5 minutes can remove microbubbles, the stirring speed of 200-250r / min balances the dispersion effect and enzyme activity protection, and the 4℃ refrigeration temperature takes into account both antibacterial properties and system stability. These process parameters have all been optimized and determined to avoid performance degradation caused by improper parameters.

[0059] The reagents prepared in this invention are compatible with fully automated biochemical analyzers and can be used for early screening, disease monitoring, and treatment efficacy evaluation of kidney diseases such as diabetic nephropathy and hypertensive nephropathy. They are particularly suitable for large-scale screening and accurate diagnosis of high-risk populations, with a testing speed of up to 300 samples per hour. Compared to existing technologies, its anti-interference rate is ≤2.5%, its attenuation rate at room temperature for 6 months is ≤4.0%, its attenuation rate at refrigeration for 12 months is ≤5.0%, its coefficient of variation is ≤1.5%, and its recovery rate is 96%-104%, demonstrating significantly superior performance across all core indicators. Furthermore, the reagents are made from biocompatible materials, are non-toxic and non-irritating, and the waste liquid can be treated according to standard medical procedures. Discarded reagent bottles can be recycled or disposed of according to medical solid waste regulations after cleaning and disinfection, meeting environmental and safety requirements.

[0060] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A preparation process for an interference-resistant dual-colorimetric detection reagent for urinary microprotein and urinary creatinine, characterized in that, Includes the following steps: (1) Buffer preparation: Dissolve the composite buffer in deionized water at 20-38℃ and stir to dissolve, and adjust the pH of the system to 7.0-7.3; (2) Addition of functional components: Add the compound anti-interference agent, EDTA-2Na masking agent and enzymes in sequence, stir continuously, and control the system temperature to not exceed 38℃; (3) Preparation of colorimetric system: Prepare the modified BCA colorimetric agent and copper sulfate auxiliary colorimetric agent separately, mix them and let stand; (4) Combination and optimization: Add the colorimetric system to the buffer system, add the combination stabilizer, stir and let stand; (5) Purification and dispensing: Before filtration, ultrasonic degassing is performed. After filtration, the product is aseptically dispensed, vacuum sealed, and then refrigerated for later use. The composite anti-interference agent contains at least two of ascorbic acid oxidase, bilirubin oxidase, and hemoglobin oxidase. The composite buffer is a combination of two different buffers.

2. The preparation process according to claim 1, characterized in that, The compound anti-interference agent is a combination of ascorbic acid oxidase and bilirubin oxidase, a combination of bilirubin oxidase and hemoglobin oxidase, or a combination of ascorbic acid oxidase, bilirubin oxidase and hemoglobin oxidase.

3. The preparation process according to claim 1, characterized in that, The composite buffer is a combination of Tris-HCl and HEPES, or a combination of HEPES and PIPES, with the molar ratio of the two buffers in the combination being 3:2 to 7:

3.

4. The preparation process according to claim 1, characterized in that, The enzymes include sarcosine oxidase and peroxidase. The dosage of sarcosine oxidase is 0.15-0.2 g / L, and the dosage of peroxidase is 0.2-0.25 g / L.

5. The preparation process according to claim 1, characterized in that, The volume ratio of the modified BCA colorimetric reagent to the copper sulfate auxiliary colorimetric reagent in the colorimetric system is 6:1 to 11:

1.

6. The preparation process according to claim 1, characterized in that, The composite stabilizer is a combination of glycerol and bovine serum albumin, a combination of glycerol and sorbitol, or a combination of bovine serum albumin and trehalose.

7. The preparation process according to claim 1, characterized in that, The ultrasonic degassing process uses a power of 100W and a processing time of 5-10 minutes.

8. The preparation process according to claim 1, characterized in that, The stirring speed in step 2 is 200-250 r / min, and the stirring time is 30-45 min.

9. The preparation process according to claim 1, characterized in that, In step 3, the standing time for the color development system is 1.5-2 hours, and in step 4, the standing time for the composite system is 1.5-2.5 hours.

10. The preparation process according to claim 1, characterized in that, The filter membrane used for filtration is a polyethersulfone filter membrane, and the refrigeration temperature after vacuum sealing is 2-6℃.

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